Allelic origin of the abnormal prion protein isoform in familial prion diseases.

The hallmark of prion diseases is the presence of an aberrant isoform of the prion protein (PrP(res)) that is insoluble in nondenaturing detergents and resistant to proteases. We investigated the allelic origin of PrP(res) in brains of subjects heterozygous for the D178N mutation linked to fatal familial insomnia (FFI) and a subtype of Creutzfeldt-Jakob disease (CJD178), as well as for insertional mutations associated with another CJD subtype. We found that in FFI and CJD178 subjects, only mutant PrP was detergent-insoluble and protease-resistant. Therefore, PrP(res) derives exclusively from the mutant allele carrying the D178N mutation. In contrast, in the CJD subtype harboring insertional mutations, wild-type PrP was also detergent-insoluble and likely to be protease-resistant. Our findings indicate that the participation of the wild-type PrP in the formation of PrP(res) depends on the type of mutations, providing an insight into the molecular mechanisms underlying the phenotypic heterogeneity in familial prion diseases.

Reorganization of axoplasmic organelles following beta, beta'-iminodipropionitrile administration.

beta, beta'-Iminodipropionitrile (IDPN), a synthetic compound that selectively impairs slow axonal transport, produced a rearrangement of the axonal cytoskeleton, smooth endoplasmic reticulum, and mitochondria. Immunoperoxidase staining using an antiserum to the 68,000-dalton neurofilament subunit demonstrated a displacement of neurofilaments toward the periphery of the axons of IDPN-treated rats. This change occurred simultaneously along the entire length of the sciatic nerve. Ultrastructural morphometry of the axonal organelles confirmed the peripheral relocation of neurofilaments and also showed a displacement of microtubules, smooth endoplasmic reticulum, and mitochondria to the center of the axons. The overall density of axonal mitochondria was increased, whereas those of other organelles were not significantly changed. Axons were reduced in size by 10--24%, the large axons being more affected than the small ones. The observed rearrangement of axonal organelles may be due to an effect of IDPN on microtubule-neurofilament interactions, which could in turn explain the impairment of the slow transport. Axons in IDPN intoxication are a useful model to study the organization of the axoplasm and the mechanism of axonal transport.

Morphological and biochemical changes in rat synaptosome fractions during neonatal development.

A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.

Quantitative autoradiographic study of labeled RNA in rabbit optic nerve after intraocular injection of (3H)uridine.

The distribution of labeled RNA in the optic nerve of the rabbit was studied by quantitative ultrastructural autoradiography after the intraocular injection of [(3)H]uridine. The highest density of silver grains related to [(3)H]RNA (27-40 grains/100 microm(2)) was found in glial cell perikarya; a slightly lower density was present in the glial nuclei (19-20 grains/100 microm(2)). Axons (4-5 grains/100 microm(2)) and myelin (2-3 grains/100 microm(2)) had the lowest grain densities. 74-83% of all counted grains were located outside the axons. By comparing the grain density distribution over the axon with that expected in the case of an exclusive labeling of the surrounding myelin and glial cell processes, it was concluded that the axons contained a number of grains representing [(3)H]RNA significantly higher than that expected to scatter from myelin and glial processes. Most of these grains were concentrated at the periphery of the axon and were not related to axonal mitochondria.

Protein synthesis in synaptosomal fractions. Ultrastructural radioautographic study.

A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30-50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75-77% of the total processes. 34-45% of the grains were related to processes containing ribosomes which accounted for only 4-7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.

Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study.

Beta,beta'-iminodipropionitrile (IDPN) produces a rearrangement of axoplasmic organelles with displacement of microtubules, smooth endoplasmic reticulum, and mitochondria toward the center and of neurofilaments toward the periphery of the axon, whereas the rate of the fast component of axonal transport is unchanged. Separation of microtubules and neurofilaments makes the IDPN axons an excellent model for study of the role of these two organelles in axonal transport. The cross-sectional distribution of [3H]-labeled proteins moving with the front of the fast transport was analyzed by quantitative electron microscopic autoradiography in sciatic nerves of IDPN-treated and control rats, 6 h after injection of a 1:1 mixture of [3H]-proline and [3H]-lysine into lumbar ventral horns. In IDPN axons most of the transported [3H] proteins were located in the central region with microtubules, smooth endoplasmic reticulum and mitochondria, whereas few or none were in the periphery with neurofilaments. In control axons the [3H]-labeled proteins were uniformly distributed within the axoplasm. It is concluded that in fast axonal transport: (a) neurofilaments play no primary role; (b) the normal architecture of the axonal cytoskeleton and the normal cross-sectional distribution of transported materials are not indispensable for the maintenance of a normal rate of transport. The present findings are consistent with the models of fast transport that envision microtubules as the key organelles in providing directionality and propulsive force to the fast component of axonal transport.

Neuronal and astrocytic differentiation in human neuroepithelial neoplasms. An immunohistochemical study.

Neuroepithelial neoplasms of childhood were examined immunohistochemically using antibodies against a neurofilament polypeptide and glial fibrillary acidic protein. Ninety-one cases, including 11 controls, were examined. Positively reacting cells, indicating neuronal and glial differentiation, were found in 59 of the 80 tumors. The study supports a neuroepithelial origin for medulloblastomas, central neuroblastomas, and primitive neuroectodermal tumors of childhood. The results also indicate that only a small number of the tumor cells differentiate along either neuronal or glial cell lines.

Electron microscopic localization of Alzheimer neurofibrillary tangle components recognized by an antiserum to paired helical filaments.

Paired helical filaments (PHF) are the main component of the Alzheimer neurofibrillary tangle; however, other elements, such as 10 and 15 nm straight filaments and granular amorphous components, are observed. In the present study, the localization of the antigenic determinants recognized by an antiserum to PHF fractions that does not recognize normal nervous tissue components is examined by electron microscopic immunocytochemistry. Employing the peroxidase-antiperoxidase procedure on neurofibrillary tangles in intact tissue, we found that the antiserum reacts with PHF and with granular amorphous material within the tangle. Following isolation of PHF by treatment with ionic detergent and labeling with immunogold, the antigenic determinants often occur at intervals rather than being uniformly distributed along the PHF. The distribution of the antigenic determinants recognized by this antiserum is similar to that previously observed for PHF determinants recognized by antibodies to neurofilament and microtubule-associated proteins. We conclude that PHF are heterogenous structures which contain PHF-specific as well as cytoskeleton-derived antigenic determinants.

Experimental increase of neurofilament transport rate: decreases in neurofilament number and in axon diameter.

In 2,5-hexanedione (2,5-HD)-induced axonal neuropathy, the rate of neurofilament (NF) transport increases in optic axons. To test the prediction that increases in the rate of polymer transport in any one locality of the axon lead directly to a decrease in the number of NF in that locality, NF and microtubules (MT) were quantitatively analyzed in axonal cross sections. In 2,5-HD axons the number of NF was 38% of that in control axons while the number of MT was not significantly changed; it appears that the drug treatment decreases NF number in the proximal axon regions, most directly through an increase in rate of NF transport. In those regions, the cross-sectional areas of the 2,5-HD-treated axons were 45% smaller than those of control axons; although the axons had shrunk in diameter, they retained their normal cylindrical shapes as measured by the index of circularity. Reduced internal expansive forces in the axon, working in conjunction with the normal external compressive forces, appear to reduce the radius of the axon. Quantitative analyses demonstrated that the average and the maximum lateral spacings between NF-NF, NF-MT, and MT-MT were all 30% larger in 2,5-HD-treated axons than in control axons. This suggests that polymers are relatively free to move laterally away from one another and to fill the available space within the axon. These observations are not consistent with models wherein 2,5-HD acts to crosslink the NF into an immobile network that can no longer advance within the axon. Instead, it appears more likely that 2,5-HD acts selectively on the interaction between some NF and the slow transport mechanism to increase the rate of NF transport.

Ubiquitin in motor neuron disease: study at the light and electron microscope.

Several neurodegenerative diseases, including motor neuron disease (MND), are characterized by formation of abnormal cytoskeleton-derived inclusions which contain ubiquitin (Ubq). We have studied the distribution of Ubq in 26 cases of MND with light and electron microscopic immunocytochemistry. Ubiquitin-positive inclusions were found in neurons of anterior horns in most cases of amyotrophic lateral sclerosis (ALS) but were not present in other forms of MND. Ubiquitin immunoreactivity was observed in 10-15 nm intraneuronal filaments, which were not stained by antibodies to neurofilaments, and on dense bodies of dystrophic neurites throughout the neuropil of anterior horns and pyramidal tracts. Data analysis showed a trend toward lower percentage of Ubq-positive neurons in cases with longer duration of illness or lower number of neurons. A high percentage of Ubq-positive inclusions occurred in cases with an aggressive clinical course, suggesting that ubiquitination takes place at early stages of the disease.

Molecular pathology of fatal familial insomnia.

Fatal familial insomnia (FFI) is linked to a mutation at codon 178 of the prion protein gene, coupled with the methionine codon at position 129, the site of a methionine/valine polymorphism. The D178N mutation coupled with the 129 valine codon is linked to a subtype of Creutzfeldt-Jakob disease (CJD178) with a different phenotype. Two protease resistant fragments of the pathogenic PrP (PrPres), which differ in molecular mass, are associated with FFI and CJD178, respectively, suggesting that the two PrPres have different conformations and hence they produce different disease phenotypes. FFI transmission experiments, which show that the endogenous PrPres recovered in affected syngenic mice specifically replicates the molecular mass of the FFI PrPres inoculated and is associated with a phenotype distinct from that of the CJD178 inoculated mice, support this idea. The second distinctive feature of the FFI PrPres is the underrepresentation of the unglycosylated PrPres form. Cell models indicate that the underrepresentation of this PrPres form results from the PrP dysmetabolism caused by the D178N mutation and not from the preferential conversion of the glycosylated forms. Codon 129 on the normal allele further modifies the FFI phenotype determining patient subpopulations of 129 homozygotes and heterozygotes: disease duration is generally shorter, insomnia more severe and histopathology more restricted to the thalamus in the homozygotes than in the heterozygotes. The allelic origin of PrPres fails to explain this finding since in both cases FFI PrPres is expressed only by the mutant allele. Despite remarkable advances, many issues remain unsolved precluding full understanding of the FFI pathogenesis.

A novel mechanism of phenotypic heterogeneity demonstrated by the effect of a polymorphism on a pathogenic mutation in the PRNP (prion protein gene).

Fatal familial insomnia (FFI) is a subacute dementing illness originally described in 1986. The phenotypic characteristics of this disease include progressive untreatable insomnia, dysautonomia, endocrine and motor disorders, preferential hypometabolism in the thalamus as determined by PET scanning, and selective thalamic atrophy. These characteristics readily distinguish FFI from other previously described neurodegenerative conditions. Recently, FFI was shown to be linked to a mutation in the prion protein gene (PRNP) at codon 178, which results in the substitution of asparagine for aspartic acid. As such, FFI represents the most recent addition to the growing family of prion protein-related diseases. The mutation that results in FFI had previously been linked to a subtype of familial Creutzfeld-Jakob disease (178Asn CJD). The genotypic basis for the difference between FFI and 178AsnCJD lies in a polymorphism at codon 129 of the mutant prion protein gene: 129Met 178Asn results in FFI, 129Val 178Asn in CJD. The finding that the combination of a polymorphism and a single pathogenic mutation result in two distinct conditions represents a significant advance in our understanding of phenotypic variability.

Differential APP gene expression in rat cerebral cortex, meninges, and primary astroglial, microglial and neuronal cultures.

Differential amyloid precursor protein (APP) gene expression was investigated in primary cultures of astrocytes, neurons and microglia from neonatal rat cerebral cortex as well as in meninges, and young and adult cerebral cortex tissues in order to define the possible contribution of individual CNS cell types in beta AP deposition. Meninges and neurons contained higher levels of total APP mRNA than glial cells and APP695 mRNA was abundant in neurons while glial cells and meninges contained higher levels of KPI-containing mRNAs. These results demonstrate cell-specific transcriptional and post-transcriptional regulation of APP gene expression in CNS cell types. In addition, the steady-state level of APPs in each cell type did not reflect mRNA levels indicating translational or post-translational regulation.

The amyloid percursor protein of Alzheimer disease is expressed as a 130 kDa polypeptide in various cultured cell types.

The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP-130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP-228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP-130 showed a pI of 6.2, while APP-228 failed to focus in the pH range of 4.7-7.0. Sequential extractions of cells with buffer and with Triton X-100 indicate that APP-130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP-130 and APP-228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.

Changes in axon size and slow axonal transport are related in experimental diabetic neuropathy.

In the sciatic system of rats with streptozocin (SZ)-induced diabetes, delay of axonal transport of neurofilament (NF) proteins, tubulin, and other proteins is associated with a change in axonal caliber, which increases by 40% in lumbar motor roots and decreases by 36% in tibial nerves. Since in large myelinated axons caliber is a function of the number of NF, which, in turn, is regulated by axonal transport, we studied the correlation of the number of NF and microtubules (MT) with axonal cross-sectional area in the sciatic system of SZ-treated rats to investigate whether the changes in caliber could be attributed to the impairment of transport. Despite the changes in cross-sectional area, diabetic axons in both proximal motor roots and distal tibial nerves maintained the ratios of number of NF and MT to cross-sectional area found in controls. Our findings suggest that, in rats with SZ-induced diabetes, the proximal and distal alterations of axonal caliber are an adjustment to the change in number of NF and/or MT that results from the impairment of the slow axonal transport.

Lack of glia-axon transfer of proteins in the normal optic system of goldfish.

An ultrastructural autoradiographic study of the goldfish optic tectum was carried out to determine whether proteins synthesized in glial cells are transferred into adjacent optic axons. Goldfish were injected intracranially over the optic tecta with [3H]leucine and fixed by perfusion 30 min, 4 and 24 h later. All unincorporated precursors were removed by repeated washings with fixatives, and tissue slices from the optic tecta were embedded and processed for electrom microscopy autoradiography (EMA). The densities of the silver grains were determined over optic axons and their myelin sheaths. The densities over the axons were lower than those over the myelin sheaths at all time intervals. The density of the intraaxonal grains, in absolute terms as well as relative to that of the myelin, was highest in the 4 h experiment. Analysis of the distribution of the grain densities over the myelin sheath and over concentric axonal compartments was carried out at this time interval to determine whether the grain density over the axon represented intraaxonal [3H]proteins or was only the result of grains 'scattered' from [3H]proteins located in the surrounding myelin sheaths. When the experimental distribution of the grain densities over the axon was compared with the theoretical distribution expected over the axon if only the myelin sheaths were labeled, no significant difference was found. This indicates that the silver grains present in the axons were scattered from the adjacent myelin sheath and did not represent intraaxonal radioactivity. It is therefore concluded that in our system there is not a quantitatively significant transfer of proteins between glial cells and adjacent axons.

Pathogenesis of experimental giant neurofilamentous axonopathies: a unified hypothesis based on chemical modification of neurofilaments.

This review summarizes current evidence suggesting that the pathogenetic basis of giant axonal neuropathies induced by neurotoxic chemicals involves a direct chemical modification of neurofilaments (NF) and/or related cytoskeletal proteins. Chemical modification of NF is believed to disrupt the normal cytoskeletal organization, which results in an alteration in NF transport rate and accumulation of NF at prenodal sites along the axon. The exact location at which axonal enlargements occur appears to be a continuous function, dependent on both the structure and dosage schedule of the chemical toxin. A unified hypothesis for the neuropathologic effect of the diverse spectrum of toxic chemicals known to induce giant axonopathies is presented, based on recently published data on the structure of NF protein. Neurotoxic chemicals are believed to alter the charge balance of highly ionic domains of NF proteins which are thought to enter into intermolecular coulombic interactions in forming the supramolecular cytoskeletal framework.

Distribution of proteins migrating with fast axonal transport. Their relationship to smooth endoplasmic reticulum.

The cross-sectional distribution of 3H-labeled proteins moving with the front and plateau of fast axonal transport was analyzed by quantitative electron microscopic autoradiography in sciatic nerves of rats following injection of a 1:1 mixture of [3H]proline and [3H]lysine into lumbar ventral horns. While in the front the transported proteins were uniformly distributed within the axons excluding the axolemma, which was not labeled, in the plateau 90% of 3H-labeled proteins were uniformly distributed and 10% were exclusively located at a 80-nm annulus with its outer edge at the axolemma. Ultrastructural morphometry showed that the density of smooth endoplasmic reticulum (SER) in a 160 nm wide subaxolemmal annulus was approximately twice that in the remaining axon, where it was uniform. These findings: (a) are consistent with the concept that membranous materials are released from the fast transport, move laterally and are inserted into the axolemma; (b) indicate that a portion of SER, largely located at the periphery of the axon, is not involved in fast transport.

Axonal transport of two major components of the ubiquitin system: free ubiquitin and ubiquitin carboxyl-terminal hydrolase PGP 9.5.

Ubiquitin (Ub), a stress protein thought to target abnormal proteins for degradation, is present in abnormal structures that occur in neuronal perikarya and axons of degenerative diseases including Alzheimer disease. To begin to assess the role of the Ub system in the axon, we studied expression and axonal transport of Ub and other stress proteins, as well as of Ub carboxyl-terminal hydrolase PGP 9.5, in the rat visual system in normal conditions and following heat-shock (HS). In the retina, both the constitutive and inducible forms of HSPs 70 were expressed under normal conditions, while in the superior colliculus the inducible form was detected only following HS. Ub, PGP 9.5 and HSPs 70 were transported in the axon exclusively with the slow component b (SCb), known to carry cytoskeletal and cytoplasmic proteins. The exceedingly long time needed for stress proteins to reach distant axonal locales at the rate of SCb (approximately 3 mm/day) makes it unlikely that they could contribute significantly to the stress response at those sites.